Recovery of bacitracin from



OTHER REFERENCES Johnson et a1.: In Vivo and in Vitro LaboratoryObservations on Bacitracin (paper presented at conference on AntibioticResearch, in Washington, D. C., on January 31, and February 1, 1947,under auspices of the Antibiotic Study Section of the National Instituteof Health, Bethesda, Md.), page 2.

Kuehl et al.: Science, vol. 102, pages 34 and 35 (1945).

Kuehl et al.: J. Am. Chem. 800., vol. 68, pages 1460-1462 (1946).

Cheronis: Seminircro and Macro Organic Chemistry (Crowell, New York,1942), pages 26 and 27.

Goorley: Some Chemical and Physical Properties of Bacitracin (presentedat conference on Antibiotic Research in Washington, D. C. on J anuary31, and February 1, 1947, under auspices of Antibiotics Study Section ofthe National Institute of Health, Bethesda, Md.)

Color Index, 1st edition (1924), pages 35 and .cipitating bacitracinfrom fermentation broths in the form ofa novel dye salt.

a compound fracture" of the tibia. tracin has beenfoundto be a potentantibac- Patentecl June 12, 1951 UNITED ST".?-\ ll:-. S' PATENT OFFICE-RECOVERY OFBACI'IRACIN FROM FERMENI AEIQN BROTHS Peter P. Regna,Woodclifi, N. J., and Isaiah A. Solomons, III, -Jackson-Heights,N.Y.,assignors to "Chas. 'Pfizer & 00., 1110., Brooklyn, N. Y., acorporation of New Jersey N Drawing. Application J lily 28, 1947, SerialNo. 764,246

, 6 Claims. 1

' This invention relates to the recovery of baci- :tracin'from crudeaqueous solutions such as fer "mentation broths, .and it has for itsObject to vi'provide .a'novel and improved process for this vpurpose.

Another objectof the inventionis to provide an eflicient and economicalmethod of pre- Another object is to recover bacitracin from llfermentation broths by precipitation with 1-(4- chloro-o-sulfophenyl)-5-hydroxy 3 methyl-4- (p (p -'tolylsulfonoxy) phenylazo) pyrazole, a i

dye which is also known as Polar Yellow 5G and Milling Yellow 5G.

Another object is to provide a novel and improved process for separatingbacitracin from bacitracin dye salts.

Still another object is to provide an eflicient and economicalmethod ofseparating bacitracin from bacitracin Polar 'Yellow '56- .salt.

Various other objects and advantages will be apparent as the'nature ofthe invention is more fully described.

Bacitracin, an "antibiotic, can be produced by fermentation in shallowlayers of tryptone, beef infusion, savita, or amigen broth, in'asoybeanlY digest" broth and in a syntheticmedium. in which l-glutamic acidprovides'the source of nitrogen and dextrose -the-source ofcarbohydrate.

October -12,'l945, page 3'76) from cultures of the The antibiotic isproducedby a Gram-positivesporulating bacillus of the B. subtilisgroupywhich was isolated by Johnson, Anker and Meleney-(Science,

contaminated tissue removed at operation from The baciterial agent whenapplied locally in certain surgical. infections .caused chiefly byGram-positive organisms.

A; March 8., 1947,.page 675) have found 'itto beffectiveinthe treatmentof furuncles, deep Meleney and Johnson (J. A.

and superficial abscesses, infected sebaceous cysts, etc.

It hasbeen reported'that the antibiotic can be recovered from thefermentation broth either by adsorption on charcoal, aluminum oxide,etc.,

or by extraction with butyl alcohol.

We have now discovered that bacitracin is almost quantitativelyprecipitated from fermentation brothsinthe formofa 'dyesalt by combiningthe bacitracin in the growth medium with the monosodium salt of l-(l-chloro o sulfophenyl) -5 hydroxy-3-methyl-4-(p-(p-tolylsul-'fonoxy)-phenylazo) -pyrazole, a certified color indicated as. Ext D &=CYel1ow. No, 4 bylthe Food and Drug Administration. Although the brothcan be treated with the dye at any'pl I withinpH 2 to 9 (outside theselimits of hydrogen ion concentration, the bacitracin is notvery stable),for

best results we prefer to carry out "the precipitation at pH 2. I

The microbiological assays for the determination of the antibacterialpotencies, hereinafter covering bacitracin of high antibiotic activityfrom bacitracin salts of 1-(4-chloro o-sulfophenyl) -5-hydroxy-3-methyl4 --v (p-i (p-tolylsulfonoxy) -phenylazo) -pyrazole, and isbased uponour discovery of a novel method of'accomplishing the separation ofthe'bacitracin dye salt into its two components. The, preferredtechnique for this is to dissolve the bacitracin Polar Yellow 5G salt indilute alkali, and then to extract the bacitracin dye into aqueous'butylalcohol, amyl alcohol, phenyl cellosolve, etc. The necessary conditionsfor carrying out this procedureare the following: (1) The solution ofthe dissolved baci- "tra'cin dye salt is to bekept below pH, 9, (2)

The organic solvent for the bacitracin dye salt is to be reasonablyimmiscible with water, ('3) The free acid of Polar Yellow'5G- is to" besoldble in the organic phase, even when theilatter is diluted with otherorganicsolvents for -purposes of depressing the solubility of thebacitracin, as will be described below, and (4) The acidity during thetransference of the bacitracin into water, from the mixture'oforganic'solvents, is to be maintained such that the bacitracin does notre-combinewith the free acid of Polar Yellow 5G dye in the organic phaseand-still isnot of so high an acidity as to inactivate the bacitracin.The acidity during this latter extraction can be maintained by anyorganic or inorganic "acid which serves to drive .the'bacitracinfrom:theorganic solvent into the aqueous phase.

Example 1 Six liters of a bacitracin fermentation-broth (25 U./ml;) wasadjusted topI-I.2.1 vwithedilute

1. IN A METHOD FOR RECOVERING A BACITRACIN SALT OF HIGH ANTIBIOTIC ACTIVITY FROM A CLARIFIED BACITRACIN FERMENTATION BROTH, THE STEP OF ADDING POLAR YELLOW 5G TO SAID BROTH, THEREBY PRECIPITATING THE BACITRACIN POLAR YELLOW 5G SALT FORMED. 